The primary sites of acquisition of HIV are at mucosal surfaces. Although epidemiological data suggests oral HIV transmission is infrequent, HIV can be found in oral secretions. In a preliminary study of oropharyngeal HIV shedding, we examined 70 HIV+ men from Seattle and Peru, with a median CD4 count of 356 cells/mul; 52% were receiving highly active anti-retroviral therapy (HAART). Blood and oropharyngeal swabs were taken at weekly intervals and evaluated for HIV RNA. 20 men (29%) had detectable HIV RNA (vRNA) in lingual and/or pharyngeal swabs. Generalized estimating equations were used to show that each 1.0 log10 increase in plasma VL was associated with a 0.4 log10 increase in pharyngeal VL (P<0.001), and tonsillectomy was associated with a 0.6 log10 reduction in pharyngeal VL (P = 0.007), but HAART was not (P > 0.1). Infectious HIV was isolated from the posterior oropharynx in 5 (25%) of 20 vRNA+ men. Median mucosal VL was 6.35 log10 copies/ml in culture-positive men vs. 4.68 log10 copies/ml in culture-negative men (P = 0.06). HIV was not isolated from saliva, buccal or salivary mucosa. Biopsies of palatine and lingual tonsil (n=6) revealed vRNA and p24 antigen within lymphoid follicles, despite HAART-suppressed plasma VL. vRNA+ cells were in close proximity to and migrating within the tonsil capsule. HIV vRNA was present within tonsilar lymphocytes, macrophages (Mphi) and fewer dendritic cells (DC). Mucosal CCR5+ lymphocytes were the most prevalent vRNA+ 'dim' Mphi often harbored > 10-fold more vRNA and infectious virus. An examination of oropharyngeal viruses revealed greater env gene diversity than blood viruses examined simultaneously, particularly from men receiving HAART (P = 0.004). These studies demonstrate that the oropharyx is a site of HIV expression, where Mphi appear to play an important role in maintaining high titers of replicative HIV. Pre-existing oral pathology and sexual acts that facilitate contact with the lingual-pharyngeal mucosa may be associated with increased risk of viral transmission. This proposal is devoted to defining the pathogenesis of HIV infection in the oropharynx and will focus on determining anatomic site(s) and cells that support HIV replication and factors that influence the frequency and titer of virus at mucosal surfaces. It employs state-of-the-art methodologies and describes a combination of in vitro studies, using tonsil explants to investigate the dynamics of infection, and in vivo studies to assess HIV population dynamics that may be unique to the oral cavity, including 'compartmentalization' of drug-resistant virus.